骨干提醒：Materials Tumor cells that have been treated with 8-azaguanine for 48 hours are removed from the drug and grown to Materials
主干提醒：Reagents Medium A - Pre-fusion Medium and Hybridoma Expansion Medium
大旨提示：Materials: P3X63Ag8.653 murine myeloma or YB2/0 &
- Tumor cells that have been treated with 8-azaguanine for 48 hours are removed from the drug and grown to a maximum concentration of 500,000 cells per ml.
- Rats or mice that were previously immunized and then boosted IV 72 hours prior to hybridization.
- Media: Auto-Pow with sodium bicarbonate plus non-essential amino acids, penicillin-streptomycin, L-glutamine and HT. For the serum-containing media , add 5 - 10% newborn calf serum.
- 40% PEG: Aldrich 1000. Made up in serum-free medium . The stock may be aliquoted and stored at -30°C.
- Other materials include: 96-well plates, sterile Petri dishes, conical 15 and 50 ml tubes, round bottom tubes, sterile dissection instruments, 37°C water bath, spleen crusher, 70% ethanol, syringe and needle.
- Medium A - Pre-fusion Medium and Hybridoma Expansion Medium
- Medium B - Fusion Medium
- Medium C - Hybridoma Recovery Medium
- Medium D - Hybridoma Selection Medium
- 皇家赌场号hj85，Medium E - Hybridoma Growth Medium
- PEG Solution
Materials: P3X63Ag8.653 murine myeloma or YB2/0 50% w/v PEG 1500, warmed to 37° C
Procedure Day 0
Medium:IMDM supplemented with 20% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 50 U penicillin, 50 µg streptomycin and 50 µM 2-ME in the absence or presence of HAT or HT for selection .
Preparation of Tumor Cells
- 50 ml Sterile conical tubes
- 15 ml Sterile conical tubes
- 10 ml sterile pipets
- 1 ml sterile pipets
- Pasteur Pipets, sterile
- 100 mm sterile Petri Dishes
- 96-well culture dishes
- 24-well culture dishes
- Multi-channel pipettor, 50-200 ml
- Pipet tips, sterile
- Reagent Reservoir, sterile
- Tupperware container
- Check water bath for cleanliness. Correct water volume and temperature .
- Soak the spleen crusher in 70% ethanol for 5 - 10 minutes and let it dry sterilely in the hood.
- Count the tumor cells. Begin washing the tumor cells with SFM as follows:
- Centrifuge the cells out of serum containing media in 50 ml conical tubes . Discard the supernatant.
- Carefully resuspend the cell pellet in approximately 1 ml of SFM. Transfer the cells to a new 50 ml conical tube. Avoid vigorous pipetting to reduce transferring proteins from the inside of the tube to the cell suspension. You want to transfer as little serum protein as possible into the new tube.
- Repeat the last two steps, transferring all of the tumor cells into a new tube.
- Add 30 - 50 ml of SFM to the cell suspension and centrifuge again. Discard the supernatant.
- Repeat the last step two more times.
NOTE: All washes are at 1,000 rpm for 10 minutes at 4° C using serum free media
Preparation of Spleen CellsWhile washing the tumor cells, prepare the spleen cells as follows:
- One week prior to the fusion, split myeloma cells into Medium A to ensure that they are well adapted.
- Grow up approximately 2 x 107 healthy cells, in mid-log phase, for each fusion.
Tissue Collection:1. Sacrifice animal by CO2 inhalation.2. Remove peritoneal cells from naive mouse for use as feeder cells by peritoneal lavage. Place cells on ICE.3. Sacrifice immune animal and remove spleen. Place on ICE.
- Bleed the animal for antisera and let the blood clot at room temperature for 1 - 2 hours. Transfer the blood overnight to a 4°C refrigerator before removing the clot.
- Remove the spleen and place it in a sterile Petri dish containing 10 ml of SFM. Move this Petri dish into the hood.
- Transfer the spleen with sterile forceps into new sterile Petri dish containing 10 ml SFM. This step reduces the nonsterile contaminants that may be present in the first Petri dish).
- Crush the spleen with the sterile spleen crusher. Push down hard on the entire spleen once only. Do not mash in a circular motion. If the spleen is crushed more than once or twice, fibroblasts will be released into the spleen cell mixture.
- Carefully pipet the spleen cell mixture up and down in the Petri dish to break up large cell clumps.
- Transfer the cell suspension to a 15 ml conical tube .
- Let debris settle out of the cell solution .
- Transfer clean supernatant into a new 15 ml conical tube.
- Centrifuge the cells for 5 minutes at 1200 RPM.
- Resuspend the cell pellet in 10 ml of SFM and count 5 µl on a hemocytometer.
Cell Preparation:1. Tease spleen in ice cold serum-free medium . Pass cell suspension through a Falcon 70 micron cell filter and suspend in 50 ml of ice cold I-0. Centrifuge and wash cells three times at 4° C in I-0. Resuspend cells after the third wash in 10 ml I-0 and count viable cells. Keep cells on ice.2. Concurrently with the spleenocytes, centrifuge and wash myeloma cells three times, using I-0 and resuspend in I-0. Count viable cells.3. In addition, wash peritoneal cells in I-0 twice, resuspend in I-20 and count.4. Add an appropriate number of myeloma cells to the entire volume of spleen cells according to the following ratios and centrifuge together.
Cell FusionThe ideal cell ratio of spleen to tumor cells is 5:1. Fuse a maximum of 1.5 - 2.5 x 108 spleen cells per tube. If fusing a larger number of spleen cells, divide them into two tubes. Mix the appropriate volumes of each cell suspension together and centrifuge the cells.
- Count the myeloma cells and resuspend to 2 x 107 cells in 30 ml Medium A in a 50 ml tube.
- Sacrificed the mouse, saturate in ethanol, and remove the spleen.
- Place the spleen in a Petri dish containing 10 ml of Medium A.
- Prepare a single cell suspension of the spleen.
- 融合与克隆，一般融合方法。Using a Pasteur Pipet, transfer the spleen cells to a 50 ml tube.
- Rinse the Petri dish with another 10 ml of Medium A and add to the tube.
- Allow the tube to sit for approximately 1 minute to settle the larger pieces of tissue. Transfer the cell suspension to a clean tube, leaving behind the larger pieces of tissue.
- Add 10 ml of Medium A to the tube to wash the tissue pieces. Allow to settle. Transfer the medium to the clean tube, combining it with the previous cell suspension.
- Centrifuge the splenocyte suspension at 400 g for 10 minutes, RT.
- Resuspend the cells in 10 ml of Medium A and count.
- Combine 108 viable spleen cells with 2 x 107 myeloma cells in a 50 ml tube. Centrifuge at 400g for 10 minutes.
- Discard the supernatant and wash the pellet twice with 40 ml Medium B, pre-warmed to 37oC.
- Discard the supernatant. Tap the bottom of the tube to loosen the pellet.
- Add 1 ml of PEG solution to the pellet over a 1 minute period, continually stirring the cells.
- Continue stirring for an additional 1 minutes.
- Stop the fusion by adding Medium B while constantly stirring.1 ml over 1 minute3 ml over 1 minute10 ml over 1 minute
- Incubate for 5 minutes in a water bath at 37oC.
- Slowly add 40 ml of Medium A.
- Centrifuge the cells at 400 g for 7 minutes.
- Discard the supernatant and wash the cell in 40 ml of Medium A.
- Slowly resuspend the pellet in 10 ml of Medium C.
- Transfer to a T75 flask containing 40 ml of Medium C.
- Incubate 16-24 hours at 37oC, 5% CO2.
- Thaw Medium D and mix.
- Transfer the cells from the flask into 2x50 ml centrifuge tubes and centrifuge at 400 g for 10 minutes.
- Discard the supernatants and tap to loosen the pellets.
- Combine the pellets and transfer the cells to Medium D. Mix gently by swirling the tube.
- Let sit for 30 minutes at 37oC, 5% CO2.
- Plate 9.5 ml of cells into 10-100 mm Petri dishes. Tilt the plates to level the mixture.
- Transfer the plates to a Tupperware container containing a Petri dish with 10 ml sterile water.
- Incubate plates at 37oC, 5% CO2.
Fusion Protocol:1. Aspirate all supernatant and suspend pellet by tapping the end of the tube. Place tube in container of warm water . Gradually, over a period of 30 seconds, add 1 ml of 37° C PEG while tapping the side fo the tube to achieve thorough mixing. Over the next 90 seconds continue to mix. After approximately 1 minute 40 seconds stop mixing and fill a 5 ml pipet with warm I-0. When exactly 2 minutes have elasped, dilute the PEG/cell mixture slowly by adding dropwise 1 ml of I-0 over a 1 minute time span. During the next 1 minute, add 2 ml I-0 dropwise. The remaining 2 ml I-0 in the pipet is added during the next 40 seconds. Next use a 10 ml pipet and add 14 ml 37° C I-0 during the last 1 minute period. Bring the total volume to 50 ml using I-20. Centrifuge at 4°C and resuspend in HAT medium at the appropriate volume to bring the cells to the following concentrations:
- In the water bath, bring the following to 37°C : small pop top tube containing 1 ml 40% PEG small pop top tube containing 1 ml SFM 50 ml conical tube containing 20 ml SFM empty 50 ml conical tube
- Resuspend the spleen-tumor cell pellet carefully in 0.5 ml of SFM and transfer to a 12 ml round bottom tube. Centrifuge at 700 RPM for 8 minutes to form a loose pellet. Monitor the centrifuge speed and time.
- Remove all of the supernatant from the pellet.
- Ideally the fusion will be done in a tissue culture hood that has access to containers of 37°C water. Otherwise, the fusion is done in the 37°C water bath outside of the tissue culture hood. Take particular care to keep all sterile tubes, solutions and pipets uncontaminated. Do not let water from the bottom of tubes drip into other tubes.
- Disrupt the cell pellet by flicking and/or tapping the bottom of the tube.
Perform the following steps described below. Timing is critical. Use a stop watch to keep track of the time for each step. Whenever possible, perform the procedure over the entire time period, not merely in the first few moments of the time period.
Time Procedure 00-30 seconds: Add 0.5 ml PEG; tap the bottom of the tube to mix. 30-60 seconds: Add remaining 0.5 ml; tap tube to mix. 1-2 minutes: Over this time period, slowly add 1 ml of SFM while agitating the tube. 2-6 minutes: Add 20 ml of SFM over the remaining 4 minutes. As the volume increases in the original round bottom tube, transfer the contents into an empty 50 ml conical tube.
The longer the cell solution remains in contact with the PEG, the more cells will fuse. Because PEG is toxic to the cells, it must be slowly diluted with SFM. The dilution of the cell-PEG solution should be slower in the beginning of the time course than at the end. Rumor has it that the contaminants in the PEG decrease the efficiency of fusions with different lots of PEG. These usually have an odor, therefore a quick way to screen PEG is to smell it and not use the smelliest batches.
- Centrifuge the cells out of SFM and resuspend them in serum-containing medium. For each tube of fused cells, plate the cells into 4 - 6 96-well plates at 0.1 ml per well .
- Incubate at 37°C.
Alternatively, the cells can be resuspended in I-20 2-ME with 150 µl added per well. An additional 50 µl of a 4X HAT solution can be added at 16-24 hours after fusion.
Day 1Feed the cells by adding an additional 0.1 ml per well with media containing serum and HT plus 2x methotrexate. Process and store the antisera.Day 3Replace 0.1 ml of media from each well with 0.1 ml of fresh HT media.Day 7Repeat the Day 3 procedure.Day 11Repeat the Day 3 procedure, and continue to feed every 7 days.Screen the wells when the media just begins to turn from the usual orange color to yellow. This insures that the cells have grown to a number adequate to produce detectable antibody but that they not in danger of overgrowing. The screening typically occurs between days 11 - 14.
- After 10-14 days, examine the plates for the presence of colonies.
- Remove isolated colonies from the plates using a pipettor with a sterile tips. Set the pipettor for 10 ml.
- Pipet each colony into a separate well of 96-well plates contain 200 ml of Medium E.
- Incubate the plates at 37oC, 5% CO2 for 1-4 days without feeding.
- Remove the supernatants from the wells and test. Refeed the wells with 200 ml Medium E or other Hybridoma expansion medium.
Add peritoneal cells to the fused cells at 2.5 x 104 cells per ml .
Dispense cells into 96 well plates as follows:Cells in I-20 2-ME 150 µl per well 50 µl per well 4X HAT after 18-24hrs
Recloning in ClonaCell-HYNote: More than 95% of the colonies will be monoclonal when selected by ClonaCell-HY. Recloning can be done to ensure stability.
- Once the cells are growing well in 24-well plates, resuspend the cells with a 1 ml pipet.
- Remove 10 ml of the cell suspension and add to 1.0 ml of Medium A. Mix well.
- Remove 100 ml of this suspension and add to a tube containing 10 ml of Medium D. Mix well and add to a 100 mm Petri dish.
- Spread evenly by tilting the plates. Incubate at 37oC, 5% CO2 as previously described.
- Repeat for each clone.
- After 10-14 days, select colonies and transfer to 96-well plates before testing.
The day of the fusion is considered day 0. Fusion plates are examined at 24-48 hours for any abnormalities . On day 7, wells are inspected visually and then fed. One half of the volume in each well is aspirated using a sterile pasteur pipet. A new pipet is used for each plate. Wells are fed with 125 µl of I-20 2ME HAT on days 7, 11 and thereafter as needed, i.e. Mon., Wed., Fri.
Cultures are examined visually at each feeding. Once a majority of wells appear 50% confluent for growth, supernatants are harvested for screening by the investigator. Plates are fed at this time.
**Investigators should provide information on the serum titer of the immunized animal. If the titer is greater than 1:10,000, it may be important to feed the cultures at least 3-4 times prior to the screening. This will essentially "wash away" any antibody release from dying B cells and decrease background Ig levels.